The cytotoxic effect of Clostridioides difficile pore-forming toxin CDTb

Clostridioides (C.) difficile is clinically highly relevant and produces several AB-type protein toxins, which are the causative agents for C. difficile-associated diarrhea and pseudomembranous colitis. Treatment with antibiotics can lead to C. difficile overgrowth in the gut of patients due to the disturbed microbiota. C. difficile releases large Rho/Ras-GTPase glucosylating toxins TcdA and TcdB, which are considered as the major virulence factors for C. difficile-associated diseases. In addition to TcdA and TcdB, C. difficile strains isolated from severe cases of colitis produce a third toxin called CDT. CDT is a member of the family of clostridial binary actin ADP-ribosylating toxins and consists of two separate protein components. The B-component, CDTb, binds to the receptor and forms a complex with and facilitates transport and translocation of the enzymatically active A-component, CDTa, into the cytosol of target cells by forming trans-membrane pores through which CDTa translocates. In the cytosol, CDTa ADP-ribosylates G-actin causing depolymerization of the actin cytoskeleton and, eventually, cell death. In the present study, we report that CDTb exhibits a cytotoxic effect in the absence of CDTa. We show that CDTb causes cell rounding and impairs cell viability and the epithelial integrity of CaCo-2 monolayers in the absence of CDTa. CDTb-induced cell rounding depended on the presence of LSR, the specific cellular receptor of CDT. The isolated receptor-binding domain of CDTb was not sufficient to cause cell rounding. CDTb-induced cell rounding was inhibited by enzymatically inactive CDTa or a pore-blocker, implying that CDTb pores in cytoplasmic membranes contribute to cytotoxicity.